northern blot

Euan R. Taylor etaylor at ccu.umanitoba.ca
Thu Dec 10 12:51:47 EST 1992


In <1992Dec4.075044.6195 at gserv1.dl.ac.uk> ED at micro.uct.ac.za (Ed Rybicki) writes:

>> Thanks for the northern procedure.  Do you know what really matters, ie. why do
>> es it not work for so many people?  Also, have you tried a Dig-riboprobe?  Dave
>>  Knecht


>A pleasure - since we have it working...!

>I don't know why people have problems - except maybe they're losing RNA at
>some point due to nuclease contamination of blocking buffer / washing
>buffer / whatever.  I have seen what looks like negatives (as seen by
>others on the Net) from mRNA blots probed with Dig-DNA, and I can only
>surmise that the RNA has been eaten clean off the blot.  No, we haven't
>tried riboprobes yet, but will soon.  Glad was of use!  ____________________________________________________________________
> | Ed Rybicki, PhD             |    "Now you've got the hang of it    |
> | (ed at micro.uct.ac.za)        | There's nothing you can't do with it |
> | Dept Microbiology           |        If you're very into it        |
> | University of Cape Town     |         You can't go wrong...."      |
> | Private Bag, Rondebosch     |                                      |
> | 7700, South Africa          |               -Mad John              |
> | fax: 27-21-650 4023         | (Ogden's Nut Gone Flake, Small Faces)|
>  --------------------------------------------------------------------


Thanks for the protocol and ideas , however re the "blank/negative bands":

I dont think its anything to do withh the degradation of RNA on the
filter, we have done rna and dna dot blots, and northerns, probed with
DIG-DNA we get zip, nothing, zero, reprobing the exact same blots with 32p
we get wonderful resullts.
Besides, Ihave never autoclaved either the gel components, or blotting
solutions for northerns and have never had any problem with
degradation, of either RNA on the blot or RNA probes in the hybridisation
mix. HOwevwer I confess I have only run northerns in formaldehyude gels
Formldehyde/formamide (if you use thhem) semm to stop all enzyme action,
thhe formaldehyde denatures and fixes any proteins.
On point o information, I have heard that some people actually
successfully re use their northers after RNAse wahing them, I never had
the nerve to try it myself, is thhis another of those tall stories or has
anyone out there actually done it?
The problems people are experiencing are quite definitely related to the
use of the DIG-DNA system, whether its something to with the hybridisation
oor labelling or wnhatever(it doesnt seem to be the labelling in our case
at least)
Dhere are plenty of people who replied to my earlier posting who are
having no problems with 32p and insurmountable problems with DIG. It
sounds like DIG RNA is more reliable, but then if we'd wanted to use
DIG-RNA we wouldnt hhave been mewssing around with a DIG-DNA system
The best I can say for it is that it seems to be extremely sensitive to
some (unspecified)  problem or other making it very unreliable for a LOT
of the people who have tried to use it. Boehringer don't seem to be a lot
of help

Euan

"the opinions above are my own and not those of my institution"



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